Species recognition in the truffle genus Tuber -- the synonyms Tuber aestivum and Tuber uncinatum

The two morphologically similar truffles Tuber aestivum and T. uncinatum have caused confusion because T. uncinatum is regarded by different authors, as either a distinct species, variety, subspecies, or synonym of T. aestivum. A clarification of the relationship between the two truffles would help both conservation biology and cultivation. We aimed both to test the reliability of the only quantitative morphological character used to distinguish the two taxa, i.e. the height of the spore reticulum, and to compare sequences of the ribosomal DNA (rDNA) internal transcribed spacer (ITS) region. Our study included 117 fruit bodies of T. aestivum and T. uncinatum, originating from eight European countries. The results showed that the spore reticulum height is not diagnostic. The phylogenetic analysis of ITS sequences from 81 fruit bodies and an additional 32 sequences from GenBank showed that T. aestivum and T. uncinatum were intermingled in one highly supported (100% bootstrap) monophyletic clade, separate from its sister species Tuber mesentericum. We conclude that T. aestivum and T. uncinatum are synonyms and the species should be named T. aestivum, as the oldest name has priority. For traders, T. aestivum syn. T. uncinatum should be used until conformity has been reached.     

Molecular species of ceramides from the ascomycete truffle Tuber indicum

The ceramide fractions were isolated from the chloroform/methanolic extractable of the fruiting bodies of Tuber indicum and separated into three kinds of molecular species TI-1, TI-2, and TI-3 by normal and reverse phase silica gel-column chromatography. By means of (1)H NMR and (13)C NMR spectroscopy, fast atom bombardment mass spectrometry (FAB-MS), and chemical degradation experiment, their component sphingoid base for TI-1 and TI-2 was uniformly (2S,3S,4R)-2-amino-1,3,4-octadecantriol, while the sphingoid of TI-3 was d-erythro-sphingosine, and their structures have been determined unequivocally to be (2S,2'R,3S,4R)-2-(2'-d-hydroxyalkanoylamino) octadecane-1,3,4-triol, the fatty acid composition of which consists of 2-hydroxydocosanoic, 2-hydroxytetracosanoic, and 2-hydroxytricosanoic acids (from major to minor); (2S,3S,4R)-2-(alkanoylamino)octadecane-1,3,4-triol, the fatty acid composition of which is unusual and consists of docosanoic, hexadecanoic, tricosanoic, octadecanoic and nonadecanoic acids (from major to minor); and (2S,3R,4E)-2-(alkanoylamino)-4-octadecene-1,3-diol, the component fatty acids of which were hexadecanoic (predominant) and octadecanoic acids, respectively.     

The mate recognition protein gene mediates reproductive isolation and speciation in the Brachionus plicatilis cryptic species complex

ABSTRACT: BACKGROUND: Chemically mediated prezygotic barriers to reproduction likely play an important role in speciation. In facultatively sexual monogonont rotifers from the Brachionus plicatilis cryptic species complex, mate recognition of females by males is mediated by the Mate Recognition Protein (MRP), a globular glycoprotein on the surface of females, encoded by the mmr-b gene family. In this study, we sequenced mmr-b copies from 27 isolates representing 11 phylotypes of the B. plicatilis species complex, examined the mode of evolution and selection of mmr-b, and determined the relationship between mmr-b genetic distance and mate recognition among isolates. RESULTS: Isolates of the B. plicatilis species complex have 1-4 copies of mmr-b, each composed of 2-9 nearly identical tandem repeats. The repeats within a gene copy are generally more similar than are gene copies among phylotypes, suggesting concerted evolution. Compared to housekeeping genes from the same isolates, mmr-b has accumulated only half as many synonymous differences but twice as many non-synonymous differences. Most of the amino acid differences between repeats appear to occur on the outer face of the protein, and these often result in changes in predicted patterns of phosphorylation. However, we found no evidence of positive selection driving these differences. Isolates with the most divergent copies were unable to mate with other isolates and rarely self-crossed. Overall the degree of mate recognition was significantly correlated with the genetic distance of mmr-b. CONCLUSIONS: Discrimination of compatible mates in the B. plicatilis species complex is determined by proteins encoded by closely related copies of a single gene, mmr-b. While concerted evolution of the tandem repeats in mmr-b may function to maintain identity, it can also lead to the rapid spread of a mutation through all copies in the genome and thus to reproductive isolation. The mmr-b gene is evolving rapidly, and novel alleles may be maintained and increase in frequency via asexual reproduction. Our analyses indicate that mate recognition, controlled by MMR-B, may drive reproductive isolation and allow saltational sympatric speciation within the B. plicatilis cryptic species complex, and that this process may be largely neutral.     

Species size affects hatching response to different temperature regimes in a rotifer cryptic species complex

The strategy of decreasing size with increasing temperature known as Bergmann’s rule operates at geographic scale and in spite of extensive studies still presents a puzzle to researches. The aim of this work was to verify whether patterns of diapausing egg hatching of closely related species are consistent with the expectations from Bergmann’s rule. In a laboratory study of the cryptic species complex of the rotifer Brachionus plicatilis, the response to temperature (17.5, 22.5, and 25.0 °C) of diapausing eggs collected in a pond where the three tested species coexist was assessed by (1) the number of hatchlings emerging or (2) the time needed to hatch. Our results show that the response to temperature from the sediment egg bank is species-dependent. In line with Bergmann’s rule, an association was observed between the hatching response to temperature and organism size. The biggest species increased its proportion in the hatchlings as temperature decreased, and vice versa. The intermediate size species showed an intermediate pattern. Nevertheless, temperature showed a general positive effect on hatching success. Time needed to hatch suggests a signature of Bergmann’s rule, but other factors seem to be affecting this parameter. This is the first study to demonstrate differences in the thermal dependence of diapausing egg hatching among closely related species with different body sizes, and it supports that Bergmann’s rule can be applied to diapause, an important phase in many invertebrate lifecycles.     

Niche partitioning in a sympatric cryptic species complex

Competition theory states that multiple species should not be able to occupy the same niche indefinitely. Morphologically, similar species are expected to be ecologically alike and exhibit little niche differentiation, which makes it difficult to explain the co‐occurrence of cryptic species. Here, we investigated interspecific niche differentiation within a complex of cryptic bumblebee species that co‐occur extensively in the United Kingdom. We compared the interspecific variation along different niche dimensions, to determine how they partition a niche to avoid competitive exclusion. We studied the species B. cryptarum, B. lucorum, and B. magnus at a single location in the northwest of Scotland throughout the flight season. Using mitochondrial DNA for species identification, we investigated differences in phenology, response to weather variables and forage use. We also estimated niche region and niche overlap between different castes of the three species. Our results show varying levels of niche partitioning between the bumblebee species along three niche dimensions. The species had contrasting phenologies: The phenology of B. magnus was delayed relative to the other two species, while B. cryptarum had a relatively extended phenology, with workers and males more common than B. lucorum early and late in the season. We found divergent thermal specialisation: In contrast to B. cryptarum and B. magnus, B. lucorum worker activity was skewed toward warmer, sunnier conditions, leading to interspecific temporal variation. Furthermore, the three species differentially exploited the available forage plants: In particular, unlike the other two species, B. magnus fed predominantly on species of heather. The results suggest that ecological divergence in different niche dimensions and spatio‐temporal heterogeneity in the environment may contribute to the persistence of cryptic species in sympatry. Furthermore, our study suggests that cryptic species provide distinct and unique ecosystem services, demonstrating that morphological similarity does not necessarily equate to ecological equivalence.     

Sibling species and cryptic speciation in the Brachionus plicatilis species complex (Rotifera)

An attempt is made to identify sibling species within the Brachionus plicatilis complex (Rotifera). Allozyme and morphological data for nine strains from all over the world are provided. Although the analysis of morphological data classified individuals from nine strains into two groups, cluster analysis using genetic distance data obtained from allozyme data revealed at least three groups. A male choice design is described for the first time in rotifers and was used to test for male mating preferences among sympatric strains belonging to three distinct species. The results suggest that the B. plicatilis complex is actually composed of more than three sibling species.     

Multiple cryptic species with divergent substrate affinities in the Serpula himantioides species complex

Serpula himantioides is a widespread saprotrophic morphospecies mainly colonising coniferous wood in nature, but it appears frequently in buildings as well. From an earlier study, it is known that at least three divergent lineages occur within the S. himantioides species complex. In this study, a broader sample of S. himantioides isolates has been analysed by multi-locus sequencing, including new isolates from Asia, North and South America. Altogether five phylogenetical species (PS1-5) were detected, all recognised across independent gene phylogenies. A new southern South American phylogenetic species (PS1) was found, representing an early diverging lineage within the S. himantioides species complex. The two closely related PS2 and PS3 lineages included isolates from North America only, and PS4 was also dominated by North American isolates. Most of the investigated isolates (76%) clustered into PS5, a lineage that has been found on most continents, including North America. Overall, little phylogeographical structure was found in PS5, indicating frequent and recent long-distance dispersal events within this widespread lineage. Our analyses indicate that South and North America are the centres of divergence for the S.?himantioides species complex. Some of the lineages seem adapted to various substrates, but PS5 is able to decay a wide array of angiosperms and gymnosperms, which may have facilitated the spread of this lineage throughout the world.     

Phylogeography of the Hypnea musciformis species complex (Gigartinales,Rhodophyta) with the recognition of cryptic species in the western Atlantic Ocean

Populations of the marine benthic red macroalgae Hypnea musciformis and Hypnea pseudomusciformis along the Atlantic and Pacific Oceans were tested for phylogeographic structure using the DNA barcode COI‐5P combined with rbcL for the construction of the phylogenetic tree. Strong patterns of genetic structure were detected across 210 COI‐5P DNA sequences, and 37 COI‐5P haplotypes were found, using multiple statistical approaches. Hypnea musciformis was found in the Northeast and Northwest Atlantic, the Mediterrean Sea, Namibia, and along the Pacific coast of Mexico. Two new putative species were detected, Hypnea sp. 1 in the Caribbean Sea and Hypnea sp. 2 in the Dominican Republic. Three distinct marine phylogeographic provinces were recognized in the Southern Hemisphere for H. pseudomusciformis: Uruguay, South‐Southeast Brazil, and Northeast Brazil. The degree of genetic isolation and distinctness among these provinces varied considerably. The Uruguay province was the most genetically distinct, as characterized by four unique haplotypes not shared with any of the Brazilian populations. Statistically significant results support both, isolation by distance and isolation by environment hypotheses, explaining the formation and mantainance of phylogeographic structuring along the Uruguay‐Brazil coast. Geographic, taxonomic and molecular marker concordances were found between our H. pseudomusciformis results and published studies. Furthermore, our data indicate that the Hawaiian introduced populations of H. musciformis contain Hypnea sp. 1 haplotypes, the current known distribution of which is restricted to the Caribbean.     

Improved resolution of major clades within Tuber and taxonomy of species within the Tuber gibbosum complex

Tuber gibbosum Harkn., described from northern California, originally was thought to be a single, variable species that fruited from autumn through winter to spring. It has become popular as a culinary truffle in northwestern USA, where it is commercially harvested. Morphological studies suggested it might be a complex that includes at least two species. We conducted morphological and phylogenetic studies of the complex to determine how many species it might contain and how they differed morphologically, geographically and seasonally. We also provide the first LSU phylogeny for the genus Tuber. Phylogenetic analyses resolve nine major clades in the genus with high bootstrap support and distinguish the Gibbosum clade from the Aestivum, Excavatum, Macrosporum, Magnatum, Melanosporum, Puberulum, Rufum and Spinoreticulatum clades. Further analyses of ITS and LSU regions revealed four distinct species in the Gibbosum complex. Although morphologically similar the four species differ in spore size and shape and in peridial anatomy. These species share the synapomorphy of having suprapellis hyphae with distinctive, irregular wall swellings at maturity; we have not seen this hyphal type in any other Tuber spp. worldwide. The three new species are named and described as T. bellisporum Bonito & Trappe, T. castellanoi Bonito & Trappe and T. oregonense Trappe, Bonito & Rawlinson.     

Ribosomal DNA-probes differentiate five cryptic species in the Anopheles gambiae complex

This study describes the use of ribosomal DNA probes to identify the species of individual mosquitoes in the Anopheles gambiae complex, a group of six morphologically identical mosquito species among which are two of the principal vectors of malaria in Africa. The DNA probes are sequences of DNA derived from the ribosomal genes of An. gambiae. Each probe reveals a different sized restriction enzyme fragment specific to each of the five species in the complex that were examined in this study: An. gambiae, An. arabiensis, An. quadriannulatus, An. melas and An. merus. The probes detect highly repeated sequences of DNA, thus the method is sufficiently sensitive to be applied to a small portion of a mosquito. Furthermore, because the DNA can be extracted from desiccated or alcohol preserved specimens, the test is compatible with other mosquito assays performed on dried specimens such as blood meal and malaria sporozoite antigen ELISAs. Determination of the nucleotide sequences that underlie the species-specific restriction enzyme site differences detected by these probes will lead to the development of synthetic DNA probes that can be used to identify an individual mosquito to species on the basis of a simple dot-blot or squash-blot.     

印度块菌提取物抗氧化活性的研究

对印度块菌Tuber indicum子实体的提取物,包括55%乙醇粗提物(ECE)、石油醚提取物(PEF)和乙酸乙酯提取物(EAF)的清除DPPH自由基和羟基自由基能力、铁离子鳌合能力以及各提取物的总酚含量等进行了研究和测定,结果显示,3种提取物的清除自由基能力和铁离子鳌合能力具有显著差异(P0.05);ECE对DPPH自由基的清除活性最高,其EC50值为1.61g/L;EAF对羟基自由基及铁离子表现出较强的清除或螯合的能力,其EC50值分别为3.31g/L和0.70g/L;EAF的总酚含量(2.964mg GAE/g提取物)最高,其次是ECE,总酚含量为(2.618mg GAE/g提取物);PEF的清除自由基和铁离子鳌合能力较差,其总酚含量也最低(1.124mg GAE/g提取物);总酚含量与印度块菌提取物清除自由基以及鳌合铁离子的能力密切相关。     

Phenotypic similarity and differentiation among sympatric cryptic species in a freshwater amphipod species complex

1. Cryptic species diversity poses evolutionary questions about its origin and maintenance, and ecological questions about the coexistence of seemingly identical species. 2. We examine patterns of morphological and life history differentiation in three sympatric cryptic species of freshwater amphipods within the Hyalella azteca species complex. These species are separated by extensive molecular evolution, but appear similar in phenotype. Species were collected from the littoral zone of a small kettle lake in Michigan, U.S.A., and identified to species by molecular genetic methods. 3. Two of the species were similar, differing only in female size, whereas the third was larger in body size and had larger clutches of smaller eggs than the other two. There were differences between the species also in pleon spine length and antennal segment number. 4. An analysis of allozyme variation among the cryptic species in three lakes suggests that the species are reproductively isolated within lakes. 5. We suggest that phenotypic similarity of these species is maintained by size‐selective predation by fish. The small, but statistically significant, differences in body size may form the axis for a tradeoff between resource exploitation and predator avoidance, a condition that can foster coexistence of phenotypically similar species.     

Isozyme variation and differentiation of morphologically cryptic species in the Brachypodium distachyon complex

Isozyme variation of nine enzymes among three morphologically cryptic species of the Brachypodium distachyon complex was studied with PAGE. Aspartate aminotransferase isozymes AAT-A and AAT-C, aryl alcohol dehydrogenase AAD-B, malate dehydrogenase MDH-A and leucine aminopeptidase LAP-A in the allotetraploid B. hybridum display fixed heterozygosities with codominant expression of homoeozymes corresponding to divergent orthozymes of diploids B. distachyon and B. stacei. The multilocus isozyme genotype inherent to most accessions of B. hybridum combines two divergent multilocus isozyme genotypes characteristic of most accessions of B. distachyon and B. stacei. The maternally inherited chloroplast-encoded RUBISCO isozymes show that B. stacei is a maternal progenitor of B. hybridum, whereas the nuclear-encoded isozymes AAD-B, AAT-A, AAT-C, LAP-A, and MDH-A confirm that B. distachyon is the paternal progenitor. These data provide a new isozyme-based support to the karyological and DNA marker data on the allotetraploid nature of B. hybridum and its origin from diploids B. distachyon and B. stacei.     

Morphological analysis of some cryptic species in the Acanthocyclops vernalis species complex from North America

Patterns of morphological variation and reproductive isolation were examined for several North American populations of copepods in the Acanthocyclops vernalis Fischer A., 1853 (Copepoda, Cyclopinae) species complex. The copepods were collected from six sites in Wisconsin, U.S.A. Morphological analysis of 120 adult females revealed that a character used previously to distinguish species in this group was unreliable because of phenotypic plasticity. Most of the morphological variance was due to environment (Laboratory vs. field) and to field site. Relatively little of the variation was due to measurement error or asymmetry. Multivariate ordination analysis produced poorly-defined clusters of individuals, suggesting that different biological species are difficult or impossible to distinguish using a set of easily-measurable morphological characters. In our study, morphological similarity was independent of geographic distance among sites, between 0.05 and 300 km. Isofemale lines within sites showed little or no reproductive isolation, but nearly complete isolation among sites. Reproductive isolation was also independent of morphology. These results suggest that the Acanthocyclops population at each site could be considered a distinct cryptic biological species. These copepods expressed morphological stasis – persistence of morphological uniformity despite reproductive isolation. Because of the effect of site and environment on morphology, we recommend using much larger collections (many sites), common garden experiments, and a multi-disciplinary approach (morphological, reproductive, chromosomal, and molecular) as the basis for future taxonomic research on putative copepod species.     

Taxonomy of cryptic species in the Cyornis rubeculoides complex in the Indian subcontinent

Taxa classified as subspecies may in fact be cryptic species. We assessed the taxonomic status of the Blue-throated Flycatcher Cyornis rubeculoides complex in India, which consists of several forms with similar plumages and song. We used mitochondrial and nuclear DNA, plumage traits, and detailed song analysis to ascertain the taxonomic status of the different forms. The molecular data identified three primary clades: (1) in the west Himalayan foothills, (2) at higher elevations in the northeast hill states of Meghalaya, Nagaland and Mizoram, and (3) at lower elevations in the northeastern hills of Meghalaya and the east Himalayas of Arunachal Pradesh. The western clade represents nominate C. rubeculoides rubeculoides. The high-elevation eastern clade was considered to be C. rubeculoides rogersi, because it included a sample from this subspecies from near the type locality in southwest Myanmar. These two sister clades had an estimated divergence time of 1.5 million years (my). The low-elevation east clade has previously been assigned to C. rubeculoides, but we showed it is closely related to the Hainan Blue Flycatcher Cyornis hainanus, formerly thought to breed only further east, with an estimated divergence time of only ~0.8 my. This clade may represent a subspecies of C. hainanus or, given reports of widespread sympatry with C. hainanus in Thailand, a distinct species, Cyornis dialilaemus. However, more research is advocated, including molecular data, from the area of overlap. Songs were remarkably similar across all taxa. In playback experiments, C. r. rubeculoides in the west responded to all taxa. This is in agreement with recent work demonstrating that song differences and responses to songs are not always a good indicator of the progress of reproductive isolation.     

Morphological recognition of cryptic species in the planktonic foraminifer Orbulina universa

One of the key hypotheses of paleoceanography is that planktonic foraminiferal morphospecies record reasonably stable and homogeneous oceanographic and climatic characteristics over their geographic and stratigraphic ranges. The discovery of numerous genetically-defined cryptic species challenges the morphospecies concept in planktonic foraminifera and paleoceanographic interpretations based on them. Here, we present a combined genetic and biometric analysis of Orbulina universa specimens in the Atlantic, Indian and Pacific Oceans. Our study is based on shells retained after DNA extractions. On those genotyped shells, we perform biometric analyses (shell size and thickness, inner porosity and pore surface distribution). Our genetic data confirm the presence of three cryptic species of O. universa in the world ocean, whose distributions are primarily correlated to the productivity of the surface waters. The Mediterranean species of O. universa is most abundant in the vertically mixed and nutrient-rich areas of the low to mid-latitudes, whereas the Caribbean and Sargasso species occur in stratified and oligotrophic subtropical waters. Our biometric data show no correlation between shell size and inner porosity within each cryptic species of O. universa. Combining Principal Component Analyses with MANOVAs performed on shell pore surface distribution, we demonstrate that the three different cryptic species are characterized by significant morphological differentiation. The Caribbean species typically exhibits large pores and higher porosity values, while the Mediterranean and Sargasso species are characterized by smaller pore areas and shell porosity. A model based upon pore surface distribution correctly assigns 60% to 90% of the specimens to their corresponding genotype. Although the inner shell surface of the Sargasso species resembles that of the Mediterranean species, our model demonstrates that the pore surface distributions of these two cryptic species can be distinguished. Finally, the Sargasso species exhibits significantly thinner shells than the two other cryptic species.     

An analysis of species boundaries and biogeographic patterns in a cryptic species complex: the rotifer--Brachionus plicatilis

Since the advent of molecular phylogenetics, there is increasing evidence that many small aquatic and marine invertebrates--once believed to be single, cosmopolitan species--are in fact cryptic species complexes. Although the application of the biological species concept is central to the identification of species boundaries in these cryptic complexes, tests of reproductive isolation do not frequently accompany phylogenetic studies. Because different species concepts generally identify different boundaries in cryptic complexes, studies that apply multiple species concepts are needed to gain a more detailed understanding of patterns of diversification in these taxa. Here we explore different methods of empirically delimiting species boundaries in the salt water rotifer Brachionus plicatilis by comparing reproductive data (i.e., the traditional biological species concept) to phylogenetic data (the genealogical species concept). Based on a high degree of molecular sequence divergence and largely concordant genetic patterns in COI and ITS1, the genealogical species hypothesis indicates the existence of at least 14 species--the highest estimate for the group thus far. A test of the genealogical species concept with biological crosses shows a fairly high level of concordance, depending on the degree of reproductive success used to draw boundaries. The convergence of species concepts in this group suggests that many of the species within the group may be old. Although the diversity of the group is higher than previously understood, geographic distributions remain broad. Efficient passive dispersal has resulted in global distributions for many species with some evidence of isolation by distance over large geographic scales. These patterns concur with expectations that micro-meiofauna (0.1-1mm) have biogeographies intermediate to microbial organisms and large vertebrates. Sympatry of genetically distant strains is common.     

Genetic diversity of a Botrytis cinerea cryptic species complex in Hungary

Botrytis cinerea has been described as a species complex containing two cryptic species, referred to as groups I and II. The first B. cinerea group I strains outside of Western Europe were collected in Hungary in 2008 from strawberry and rape plants. Sympatric B. cinerea cryptic species were analyzed using a population genetic approach and phenotypic markers. Statistically significant, but moderate population differentiation was found between the two groups in Hungary. Group I was originally typified by the lack of the transposable elements Boty and Flipper. However, all the Hungarian group I isolates carried the Boty element and one isolate additionally contained Flipper, indicating a much wider genetic variation than previously believed. Vegetative compatibility analyses showed that twelve of the thirteen B. cinerea group I isolates studied belonged to a unique vegetative compatibility group (VCG), but VCGs overlapped between groups. Phenotypic markers such as fenhexamid resistance or asexual spore size were found unsuitable to differentiate between the cryptic species. The results did not confirm the complete separation of the two cryptic species, previously determined with genealogical concordance of the phylogenetic species recognition using multiple gene sequences, and suggest instead the possibility of information exchange between them.     

国产松露乙醚提取物的GC-MS分析

采用气相色谱-质谱(GC-MS)法分析我国云南产黑松露(Tuber indicum)乙醚提取物的化学成分.从中鉴定了56个化合物,占提取物总含量的87.37％,化合物组成特征:以脂肪酸类化合物为主,占59.98％,其中含亚油酸达26.51％;醇、酯、醛和烃类化合物分别占7.96％、2.79％、2.44％和13.12％,另外还含有少量的苯衍生物,占1.08％.